Cartilage injury is common which can lead to progression of osteoarthritis (OA) which is characterized by progressive breakdown of articular cartilage, and ultimately leads to functional failure of synovial joints. Regeneration of cartilage has been an attractive approach to cartilage repair and treatment of OA. However, autologous chondrocyte transplantation suffers from insufficient cell supply, new damage to the donor sites and chondrocytes dedifferentiation during in vitro expansion. Mesenchymal stem cells (MSC) which reside in the bone marrow and many adult tissues are capable of self-renewal and differentiation into chondrocytes and are a potential source for cartilage in situ regeneration. Microfracture induces migration of bone marrow MSC to the site of cartilage defect and promotes fibrocartilage production. However, fibrocartilage is non-durable and functionally inadequate in the long-term. SOX9 is a transcription factor belonging to the SOX (Sry-type HMG box) family and has been identified as a master regulator of the chondrocyte phenotype. We have generated a cell penetrating, super positively charged SOX9 fusion protein (scSOX9). In vitro studies have demonstrated that scSOX9 protein induced bone marrow derived MSC proliferation and differentiation into chondrocytes. This was evident by decreased production of collagen type I and type X but increased collagen type II and aggrecan, which are characteristics of articular chondrocytes. In a 21 day culture, the initial one time addition of scSOX9 was sufficient to differentiate MSC into chondrocytes and maintain the chondrocyte phenotype. Preliminary in vivo data demonstrated scSOX9 significantly improved the outcome of microfracture in cartilage repair in short-term. In this proposal, we aim to promote cartilage in situ regeneration by stimulating bone marrow MSC in a rabbit model of cartilage defect. Experiments are designed to administer scSOX9 protein at the site of microfracture of the defective cartilage. Administration of scSOX9 will be carried out by either direct delivery with a bioscaffold, collagen membrane at the site of microfracture. Regeneration of cartilage will be assessed at 12 and 24 weeks using histology and immunohistochemistry for characters of hyaline cartilage by examination of content of aggrecan and collagen type I, II and X and osteocalcine. The biomechanical properties of the repaired cartilage will also be evaluated. Immune response to scSOX9 will be assessed by measuring serum antibodies and T cells reaction to scSOX9. Efficacy of re-administration of scSOX9 with microfracture will be assessed in animals who received scSOX9 previously in contralateral knee. Molecular mechanism for which scSOX9 mediated hyaline cartilage repair will be investigated. The research plan takes advantage of the minimal invasive, simple and low cost microfracture to attract bone marrow MSC and pro-chondrogenic property of scSOX9 to regenerate articular cartilage in situ for therapy of cartilage defect. The success of this project will provide invaluable information for design of a clinical trial using the same techniques for therapy of cartilage lesions and prevention of progression to OA.